Inhibiting ferroptosis mitigates sheep sperm freezing damage

Area of Science:

• Reproductive Biology
• Cell Death Pathways
• Cryopreservation Science

Background:

• Cryopreservation of sheep sperm can induce cellular damage, involving programmed cell death mechanisms.
• Understanding the roles of apoptosis and ferroptosis is crucial for improving sperm preservation techniques.
• Previous studies suggest that oxidative stress contributes to sperm damage during cryopreservation.

Purpose of the Study:

• To investigate the involvement of apoptosis and ferroptosis in sheep sperm damage induced by cryopreservation.
• To evaluate the efficacy of specific inhibitors targeting apoptosis and ferroptosis pathways in mitigating cryopreservation damage.
• To assess the impact of these inhibitors on sperm quality parameters and key molecular markers.

Main Methods:

• Compared expression of apoptotic (Cleaved-caspase3) and ferroptotic (TFRC) markers in fresh versus cryopreserved sheep sperm.
• Applied inhibitors: Deferoxamine Mesylate (DFO), ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), and Z-VAD-FMK (Z-VAD) at varying concentrations.
• Assessed post-thaw sperm viability, plasma membrane integrity, acrosome integrity, reactive oxygen species (ROS), lipid peroxidation, Fe²⁺ levels, and Glutathione Peroxidase 4 (GPX4) expression.

Main Results:

• Cryopreservation increased Cleaved-caspase3 and TFRC expression, indicating concurrent apoptosis and ferroptosis.
• Optimal inhibitor concentrations (2 μM for DFO, Fer-1, Lip-1; 5 μM for Z-VAD) significantly improved sperm motility and membrane integrity.
• Fer-1 was most effective, reducing ROS, lipid peroxidation, and Fe²⁺, and restoring GPX4 expression to fresh sperm levels.

Conclusions:

• Both apoptosis and ferroptosis are significant contributors to cryopreservation-induced damage in sheep sperm.
• Ferrostatin-1 (Fer-1) effectively mitigates ferroptosis, enhancing sperm quality and restoring GPX4 levels post-cryopreservation.
• Targeting ferroptosis represents a promising strategy for improving sperm cryopreservation outcomes, benefiting animal breeding and conservation efforts.